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At least 19 recordsLinked to original sources
PubMed1988

Adhesion patterns and cytoskeleton of rabbit osteoclasts on bone slices and glass.

The ability of osteoclasts (OC) to migrate and resorb bone is thought to be dependent on cytoskeletal function and adhesion. Therefore, we investigated the cytoskeleton and the adhesion patterns of rabbit OC on glass and on devitalized bone slices, using specific antibodies to cytoskeletal elements and fluorescence and interference reflection microscopy. Microtubules (MT) were similar in OC on both substrata, and appeared in a pattern typical of that described for many cells. Multiple centriolar complexes were observed in most OC, either as one large aggregate in the center of the cell or dispersed singly or in small aggregates close to individual nuclei. Staining of microfilaments (MF) was similar on both substrata and appeared primarily as an F-actin network. MF distribution was different in OC associated with resorption lacunae with intense staining over those regions. In the OC on glass, high F-actin staining was detectable at the periphery in dots and rosette-like structures, which also stained for vinculin. The adhesion patterns indicated that OC on glass do not make large focal contacts, but appear to make a few tiny focal contacts that are not associated with the rosette-like structures. Most of the undersurface of the OC appeared either to be involved in close contacts or to be separated by distances of greater than 100 nm from the substratum. These studies indicate that the MF distribution and the adhesion patterns of rabbit OC are typical of motile cells, that the distribution of the cytoskeleton of rabbit OC on glass and on bone slices is similar, and that MF may be involved in the morphological changes associated with resorption.

Actin Cytoskeleton↗
PubMed1990

The quantitative study of the orientation of collagen in compact bone slices.

We describe and validate a simple method for the study of the proportion of collagen fibers (and apatite crystals) in a bone slice parallel with the plane of section. Viewed between crossed circular polarizers, all bone areas with collagen lying more nearly in the plane of section (i.e., transverse [TS] collagen) appear bright whatever its direction in this plane; longitudinal [LS] collagen appears dark, but not as dark as the background. The degree of brightness increases with section thickness, which must therefore be standardized--we chose 100 microns plane parallel sections. We transferred the circularly polarized light [CPL] image via a CCD TV camera to an image analyzing computer. Color-coded maps of the CPL image were used to compare regions within and between sections. The new analytical procedure makes more detailed studies of the fine-structural orientation in compact bone possible, but does this have any significance? To answer this question, a bone in which the in vivo strain pattern had been clearly documented was chosen for particular study. Transverse mid-diaphyseal sections of the equine radius showed a distribution of CPL bright areas which correlated closely with previously reported strain patterns.

Animals↗
PubMed1990

The effects of parathyroid hormone (PTH) and PTH-related peptide on osteoclast resorption of bone slices in vitro: an analysis of pit size and the resorption focus.

The mechanism whereby PTH, a potent stimulator of bone resorption, may under certain circumstances exert anabolic effects on bone is not known, but it is possible that it involves reduction of the size of osteoclast resorption lacunae. We have therefore made a detailed in vitro study of the effects of PTH and PTH-related peptide (PTHrP) on resorption by neonatal rat osteoclasts paying particular attention to the plan area of resorption pits. In order to distinguish between increased resorption at a particular site and increased numbers of sites, we have used an eyepiece graticule to define a focus of resorption, namely an area occupying 1/116th of the bone slice, which may contain either one or several pits. In addition we have studied the relationship between the number of pits in a resorption focus and the total area of bone resorbed at the focus. We found that PTH and PTHrP, at doses between 2 x 10(-10) M and 2 x 10(-8) M, while exerting significant stimulatory effects on bone resorption, caused a reduction in the median plan area of pits. An increase in the number of resorption foci was the primary stimulatory effect of PTH and PTHrP, occurring within 6 h in the case of PTH. However, the plan area of bone resorbed at a focus showed no significant increase, despite an increase in the number of pits per focus, because as more pits were formed at a focus, the pits were smaller, thus partially dissipating the stimulatory effect of PTH on resorption. These results are consistent with the activation of new remodeling sites by PTH in vivo. Furthermore, the formation of smaller pits under the resorptive influence of PTH may, together with the maintenance of coupling between formation and resorption, play a role in the preservation of cancellous bone recorded in cases of primary hyperparathyroidism and the anabolic effect of exogenous PTH.

Animals↗
PubMed1989

Dichloromethylenebisphosphonate (Cl2MBP) inhibits bone resorption through injury to osteoclasts that resorb Cl2MBP-coated bone.

Dichloromethylenebisphosphonate (Cl2MBP, formerly Cl2MDP) inhibits bone resorption in vivo and in vitro. The mechanism by which it inhibits osteoclastic bone resorption has not been established. To investigate this, osteoclasts were isolated from rat long bone and incubated with Cl2MBP (10(-9)-10(-5) M) on bone slices. Bone resorption was assessed as plan area resorbed after 6 and 24 h by scanning electron microscopy. Although Cl2MBP inhibited bone resorption in the first 6 h of culture, inhibition was more marked in the incubation period between 6 and 24 h. This pattern of accelerating inhibition is unlike the pattern we have observed using other resorption-inhibitors, and suggested resorption-mediated osteoclast injury. In keeping with this, we found reduced numbers of osteoclasts, and morphological features of cell injury and degeneration of osteoclasts, after incubation with Cl2MBP on bone slices. Bone seemed to be an essential component in Cl2MBP-mediated injury, since osteoclast numbers and morphology on plastic coverslips were unaffected by the bisphosphonate. Moreover, bone slices preincubated with Cl2MBP showed similar effects on resorption and morphology to cultures in in which osteoclasts on bone were continuously immersed in Cl2MBP. Neither non-resorptive cells (macrophages, UMR 106 cells), nor osteoclasts prevented from resorption by calcitonin, showed evidence of cytotoxicity after incubation on bone slices with Cl2MBP. These results suggest that even relatively high concentrations of Cl2MBP in the fluid phase do not affect osteoclasts, nor does contact with Cl2MBP-coated bone surfaces, but that injury to osteoclasts, and a consequent reduction in bone resorption, occurs when osteoclasts excavate bone surfaces upon which Cl2MBP is adsorbed.

Animals↗
PubMed1992

An assay system utilizing devitalized bone for assessment of differentiation of osteoclast progenitors.

The present study provides a novel assay system to examine the differentiation of osteoclast progenitors on devitalized bone slices. We used the population of bone cells liberated enzymatically from 14-day-old mouse embryonal calvariae as a source of osteoclast progenitors. The analysis of differentiation of osteoclast progenitors into preosteoclasts and mature osteoclasts was assessed in terms of the formation of TRAP-positive cells and pits or resorption lacunae, respectively, on devitalized bone slices. Osteoclasts having bone-resorbing activity appeared when the calvarial cell population was cultured in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. The resorbing activity increased in a 1 alpha,25-(OH)2D3 dose-related manner. However, calcitonin, a potent inhibitor of differentiation and activation of osteoclast lineage cells, reduced the area of the resorption lacunae in a dose-dependent fashion. The bone-resorbing cells on the bone slices expressed an obvious ruffled border and clear zone, structures specific to mature osteoclasts. These results suggest that osteoclast progenitors in the mouse calvarial population examined differentiated into mature osteoclasts in the presence of 1 alpha,25-(OH)2D3 on devitalized bone slices. Further, using this assay system we assessed the effect of some other osteotropic factors on the differentiation of osteoclast progenitors to mature osteoclasts. IL-1, IL-6, and PTH increased the formation of TRAP-positive cells and pits and the area of resorption lacunae in a dose-dependent fashion. However, prostaglandin E2 was unable to induce the formation of resorption lacunae, although a significant appearance of TRAP-positive cells was observed at a concentration of 200 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid Phosphatase↗
PubMed1990

An in vivo model system for the study of avian osteoclast recruitment and activity.

We have developed a model system for the study of osteoclast recruitment and activity using devitalized bovine cortical bone slices implanted onto the chorioallantoic membrane (CAM) of chicken embryos. Bone slices were examined after 3, 6, and 8 days of incubation on the CAM. A marked cellular reaction to the bone was observed, characterized by a prominent angiogenic response. Upon histological examination, numerous multinucleated giant cells were associated with the undersurface of the bone slice and concentrated towards its periphery. These multinucleated cells were often associated with resorption lacunae and demonstrated ruffled borders when viewed by transmission electron microscopy. Removal of the cells and examination of the bone surface by scanning electron microscopy revealed numerous resorption pits characteristic of osteoclastic activity. These pits were evident on day 3 of incubation and appeared to be more extensive by day 8. This work demonstrates that the cells recruited to such ectopically implanted devitalized bone slices are functional osteoclasts, and that this system may provide a useful model for the study of osteoclast recruitment and activity.

Animals↗
PubMed1992

Effects of heparin on osteoclast activity.

The effect of heparin on osteoclastic bone resorption was studied in vitro using the disaggregated osteoclast resorption assay. Bone resorption was assessed by counting the resorption lacunae on bone slices by light microscopy. Low concentrations of heparin (5 micrograms/ml) increased bone resorption by isolated chick and rat osteoclasts. Among other glycosaminoglycans tested at 5 micrograms/ml, only dextran sulfate showed a small but significant stimulation of resorption. Chondroitin sulfates A, B, and C were without effect at 25 and 100 micrograms/ml, whereas resorption was increased by 100 micrograms/ml of heparan sulfate. With chick osteoclasts, which could be maintained in serum-free conditions, a stimulatory effect of heparin was found both in the presence of 5% fetal calf serum and in serum-free media containing insulin, transferrin, and selenium. The magnitude of the heparin-induced increase in resorption was similar in the presence or absence of serum. The stimulation of resorption was associated with an increase in the number of osteoclasts on bone slices. Pretreatment of the bone slices with heparin also enhanced resorption. In time course experiments, 5 micrograms/ml of heparin caused a doubling of chick osteoclast activity index (number of resorption pits per number of osteoclasts) at 12 and 24 h. In 24 h cultures, treatment with 10 micrograms/ml of the arginine-rich basic protein, protamine, 1 microgram/ml of the immunosuppressant, cyclosporine A, or 5 micrograms/ml of the cysteine-proteinase inhibitor, leupeptin, negated the heparin effect on bone resorption. Leupeptin also inhibited basal resorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Optimal bone resorption by isolated rat osteoclasts requires chloride/bicarbonate exchange.

We have examined the effect of DIDS (4,4'-diisothiocyanatostilbene sulfonic acid) a potent, specific and irreversible inhibitor of chloride/bicarbonate exchange on bone resorption by disaggregated rat osteoclasts, using an in vitro bone slice assay. DIDS inhibited bone resorption in concentration dependent fashion, without affecting osteoclast viability or survival on bone slices. The role of anion exchange in the resorptive process is discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1991

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: light and electron microscopic study.

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

Adult↗
PubMed1991

Inhibition of bone resorption by bisphosphonates: interactions between bisphosphonates, osteoclasts, and bone.

Bisphosphonates are nonbiodegradable pyrophosphate analogues that are being used increasingly to inhibit bone resorption in disorders characterized by excessive bone loss. We have previously found that dichloromethylene bisphosphonate (Cl2MBP) inhibits bone resorption through injury to the cells that resorb Cl2MBP-contaminated surfaces. 3-amino-1-hydroxypropylidene-1,1-bisphosphonate (AHPrBP) is a more potent inhibitor of bone resorption in vivo, and we have attempted to identify a step in the resorptive pathway that accounts for this increased potency. We found that when osteoclasts, isolated from neonatal rat long bones, were incubated on bone slices in the presence of bisphosphonates, AHPrBP was less, rather than more potent as a resorption-inhibitor than Cl2MBP. The greater sensitivity of resorption to AHPrBP in vivo could neither be attributed to an effect of AHPrBP on the ability of osteoblastic cells to stimulate resorption in response to calcium-regulating hormones in vitro nor to an effect on osteoclast generation: osteoclast formation was unaffected by concentrations of AHPrBP 10-fold higher than those of Cl2MBP which inhibit bone resorption in the bone slice assay. We also found no evidence for impaired osteoclast generation in vivo in AHPrBP-treated rats. These results suggest that the comparisons of potency in vitro do not include all the factors responsible for determining bisphosphonate potency in vivo. Because bisphosphonates owe the specificity of their actions to their ability to bind to bone surfaces, we performed experiments using bone slices that had been immersed in bisphosphonates before use. Bone resorption was virtually abolished on bone slices preincubated in 10(-3) M AHPrBP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Time course of "escape" from calcitonin-induced inhibition of motility and resorption of disaggregated osteoclasts.

The reversible calcitonin (CT)-induced inhibition of osteoclastic activity has been studied to clarify the mechanisms responsible for the so-called "escape phenomenon." Osteoclasts disaggregated from neonatal rabbits were cultured on glass coverslips or thin bovine bone slices. Resorption activity was evaluated by using time-lapse recording and scanning electron microscopy. Addition of CT to the cultures caused most osteoclasts on glass surfaces to be immotile and contracted. From 1.5 h onward, in cultures with CT, osteoclasts started to escape from CT-induced quiescence independently of other cells. CT also prevented osteoclasts on bone slices from excavating bone while concomitant cell immobility occurred. Inhibited osteoclasts were able to regain apparent bone-resorbing potency only after resumption of cytoplasmic immobility. The resumption of bone resorption could begin as early as 9.7 h after CT addition. The observations indicate that CT-induced inhibition of osteoclastic bone resorption is associated with inhibition of cytoplasmic motility and that the "escape" phenomenon reflects resumption of activity of osteoclasts that were previously inhibited by CT action rather than the resportive activity of newly formed osteoclasts.

Animals↗
PubMed1991

Identification of osteoclast precursors in multilineage hemopoietic colonies.

The osteoclast is known to be derived from the hemopoietic stem cell, but its lineage and the mechanisms by which its differentiation is regulated are largely unknown. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hemopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semi-solid medium from mouse spleen cells in the presence of erythropoietin with either Wehi 3B-conditioned medium or interleukin 3 (IL3). After 7 days, individual colonies were picked. Half of each colony was phenotyped by the morphology of cells in cytospin preparations; the second half of each was incubated for 7 days with a bone marrow-derived cell line (ts8) that induces osteoclastic differentiation from hemopoietic cells, on bone slices in the presence of 1,25-dihydroxyvitamin D3. After incubation, bone resorption was assessed by scanning electron microscopy. No resorption was induced in cells derived from single-lineage colonies, but resorptive cells differentiated in 17% of granulocyte-macrophage (GM) colonies and 38% of multilineage colonies. Since only a minority of GM colonies contained osteoclastic precursors, this suggests that the GM colonies that contained osteoclasts were not typical GM colonies but may have been a form of multilineage colony analagous to other multilineage colonies that contain granulocytes, macrophages, and a third cell type. No resorptive cells were formed when IL3-derived colonies were incubated on bone slices without ts8 cells. The results suggest that osteoclasts are derived from a multilineage precursor, upon which IL3 acts to generate cells capable of osteoclastic differentiation, which form resorptive cells upon incubation with bone marrow stromal cells in the presence of 1,25-dihydroxyvitamin D3.

Animals↗
PubMed1987

Bone composition in the distal forearm.

Recent data have indicated that measurements of bone mass in the very distal part of the forearm is superior to more proximal measurements in identifying osteoporosis. Bone slices from the distal part of the forearm were obtained from 16 necropsies and the trabecular fraction of the total dry bone weight was measured in adjacent bone slices, 8 mm thick. Prior to autopsy bone mass at the corresponding sites was measured using a multipath single photon absorptiometric method by which scans are obtained proximal (proximal BMC) and distal (distal BMC) to the site, where the ulna and radius are 8 mm apart. The accuracy of bone measurements at the two sites was virtually similar (r = 0.98 and r = 0.94, respectively). In both areas the amount of trabecular bone increased towards the metaphysis with a trabecular/cortical ratio ranging from 10 to 60% (wt/wt). If bone composition is known it is possible to estimate rates of bone loss from the two compartments.

Adult↗
PubMed1975

Osteophytes and the osteoarthritic femoral head.

The study describes the topography, morphology and growth of osteophytes in forty femoral heads removed from patients presenting with advanced osteoarthritis of the hip. In addition to standard histological techniques, radiography of serial bone slices in vivo bone labelling with tetracycline and 32P were used. The pattern of major osteophyte formation appeared to be influenced by the direction, degree and rate of displacement of the femoral head in relation to the acetabulum; four principal patterns of growth were noted. Osteophytes form part of extensive osteogenic processes that involve bone structure in the osteoarthritic joint.

Autoradiography↗
PubMed1992

Na+/H(+)-antiporter activity is essential for the induction, but not the maintenance of osteoclastic bone resorption and cytoplasmic spreading.

We have examined the kinetics of the effects of inhibitors of the Na+/H(+)-antiporter (dimethylamiloride) and the vacuolar H(+)-ATPase (bafilomycin A1) on bone resorption by disaggregated rat osteoclasts in the bone slice assay. Bafilomycin A1 (100 nM) inhibited resorption by approximately 95%, 75%, 80% and 60% respectively, when added at t = 0, 1, 3 or 6 hr after osteoclast adherence to bone slices, during a 24 hr culture period. The incomplete inhibition by bafilomycin A1 when added after the start of incubation was presumably accounted for by resorption that had occurred prior to addition of the compound. Dimethylamiloride (100 microM) inhibited bone resorption by 80% and 65% when added at t = 0 or 1 hr after osteoclast adherence, but was without effect when added at t = 3 or 6 hr. In addition, dimethylamiloride but not bafilomycin A1 strongly inhibited osteoclast cytoplasmic spreading. The results indicate that Na+/H(+)-antiporter activity is essential for controlling intracellular pH during early activation events stimulated by the adherence of osteoclasts to mineralized bone surfaces, which lead to cytoskeletal activation, cell spreading and bone resorption.

Amiloride↗
PubMed1990

Effects of interleukin 3 and of granulocyte-macrophage and macrophage colony stimulating factors on osteoclast differentiation from mouse hemopoietic tissue.

The effects of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and interleukin 3 (IL3) on osteoclast formation were tested by incubation of murine hemopoietic cells on plastic coverslips and bone slices with GM-CSF, M-CSF, or IL3, with or without 1,25(OH)2 vitamin D3 (1,25(OH)2D3). Osteoclastic differentiation was detected after incubation by scanning electron microscopical examination of bone slices for evidence of osteoclastic excavations, and by autoradiographic assessment of cells for 1,25(OH)2D3-calcitonin (CT) binding. The differentiation of CT-receptor-positive cells preceded bone resorption, but the number that developed correlated with the extent of bone resorption (r = 0.88). M-CSF and GM-CSF substantially reduced bone resorption and CT-receptor-positive cell formation. The degree of inhibition of bone resorption could not be attributed to effects on the function of mature cells, since M-CSF inhibits resorption by such cells only by 50%, and GM-CSF has no effect. GM-CSF inhibited the development of mature function (bone resorption) to a greater extent than it inhibited CT-receptor-positive cell formation. Since CT-receptor expression antedated resorptive function, this suggests that GM-CSF resulted in the formation of reduced numbers of relatively immature osteoclasts. This suggests that it may exert a restraining effect on the maturation of cells undergoing osteoclastic differentiation in response to 1,25(OH)2D3. Conversely, IL3, which also has no effect on mature osteoclasts, by itself induced CT-receptor expression but not bone resorption; in combination with 1,25(OH)2D3 it induced a threefold increase in bone resorption and CT-receptor-positive cells compared with cultures incubated with 1,25(OH)2D3 alone. IL3 did not induce CT-receptors in peritoneal macrophages, blood monocytes, or J 774 cells. The results suggest that IL3 induces only partial maturation of osteoclasts, which is augmented or completed by additional factors such as 1,25(OH)2D3.

Animals↗
PubMed1978

Microscopic age changes in the frontal bone of the domestic rabbit.

This study reveals age related changes in the microscopic structure of the paired frontal bone of the domestic rabbit. Undecalcified ground sections were prepared from anterior frontal bone slices removed from New Zealand White rabbits ranging from birth to 24 months of age. Included were 40 females, 4 males and 6 rabbits less than 14 days old of unknown sex. The ground sections revealed both qualitative and quantitative age changes. Qualitative changes include change in bone tissue types and developmental processes, and presence or absence of primary and secondary osteones. Quantitative changes were measured by counting primary osteones in the outer table of each frontal bone half within 1.6 mm of the metopic suture. In this region, primary osteones were absent at birth, limited in number at 14 days and generally numerous between one and three months of age. In animals older than three months, numbers of primary osteones generally decreased with increasing age. No animals older than 16 months revealed primary osteones. Possible sources of variability in the relationship between observed primary osteone number and age include ability to recognize primary osteones, methods of sampling and processing bone slices, sex related differences and diseases afflicting specimen. Significance of this study includes increased knowledge of frontal bone growth and histology.

Aging↗